Kappa-casein interactions in the suspension of the two major calcium-sensitive human beta-caseins.

The possible effects of both the beta-casein (beta-CN) phosphorylation level and the kappa-CN glycosylation level on micelle formation were studied using the doubly-phosphorylated form (beta-CN-2P) and the quadruply-phosphorylated form (beta-CN-4P) of human beta-CN, along with bovine kappa-CN to compare with previous studies using the more highly glycosylated human kappa-CN. Addition of bovine kappa-CN to human beta-CN-2P, beta-CN-4P, or a 1/1 (wt/wt) mixture of the two was at kappa/beta molar ratios from 0.0 to approximately 0.6 and micelles were reconstituted by addition of Ca+2 either directly at 37 degrees C for determination of the fraction suspended or at an initial temperature of 4 degrees that was gradually increased to 37 degrees C with the change in particle size monitored by turbidity measurements. Analysis of the data indicates that the 4P form requires more kappa-CN for stabilization than the 2P form but that the mixture of the two is more like the 4P form in that lateral kappa-kappa interactions may enhance beta-kappa interactions and micelle formation. Above a kappa/beta molar ratio of about 0.2, the caseins were fully suspended into reconstituted micelles. However, micelle size decreased at a higher ratio, indicating that the kappa-CN probably occupies a surface position and may regulate micelle size by its relative abundance. A comparison with published results suggests that the higher glycosylation level of human kappa-CN may protect a larger surface area and result in smaller micelles. Changes in reconstituted micelle size with pH indicate that positively charged groups in the kappa-CN may interact with the negatively charged phosphate esters in the beta-CN moieties in addition to kappa-beta hydrophobic interactions.

The use of lithium chloride to study human milk micelles.

Various methods have been used to study the dissociation of milk micelles in attempts to determine their structure and the interactions that stabilize them. These include the addition of urea, cooling to alter hydrophobic bonding, the addition of EDTA to sequester calcium, and changes in pH to alter molecular charge. For this study, the mild chaotropic agent LiCl was added to human milk micelles, and measurements were made on the relative percentages of the six different phosphorylation levels of beta-casein (CN) at various LiCl concentrations for different lengths of time and at different temperatures. Added LiCl had little effect at 37 degrees C but caused maximal dissociation, mainly of the beta-CN species with higher phosphorylation levels, at 23 degrees C and 4 degrees C between 1 and 2 M concentration. Comparison was made with 2-M additions of NaCl, MgCl2, and KCl at 4 degrees C, with LiCl showing the only appreciable change. The results suggest that Li+ may displace Ca2+ in protein-Ca2+-protein or protein-colloidal calcium phos+ phate-protein salt bridges and that the nonphosphorylated form of human beta-CN may change its conformation and mode of interaction upon phosphorylation. Lithium chloride may be useful to study the dissociation of the different CN in bovine milk micelles.

Formation of reconstituted casein micelles with human beta-caseins and bovine kappa-casein.

Human beta-casein (CN) is the major protein of the human milk casein fraction (approximately 80%) and exists in six calcium-sensitive forms, having zero to five organic phosphates per molecule. The major forms are the doubly-phosphorylated (beta-CN-2P; approximately 30%) and the quadruply phosphorylated (beta-CN-4P; approximately 35%) forms. Although calcium-insensitive, kappa-CN is known for its role in preventing the precipitation of beta-CN in the presence of Ca+2, but it is not known how the different levels of phosphorylation may affect this. In the present investigation, turbidity, measured at 400 nm, was determined at increasing temperatures (4 up to 37 degrees C) for solutions of beta-CN-2P and beta-CN-4P (3 mg/ml in 0.02 M NaCl, 0.01 M imidazole, pH 7) individually and also mixed with bovine kappa-CN in 6/1 and 3/1 weight ratios of beta/kappa and containing 0, 5, and 10 mM Ca+2. The results indicate that the first step of micelle formation probably leads to polymers of limited size, the only complexes available to beta-CN-2P under most conditions. With beta-CN-4P, these polymers aggregate further to give reconstituted micelles, probably because of the ability to form crosslinks at this phosphorylation level. The formation of reconstituted micelles under various conditions of pH, Ca+2 concentration and kappa-CN content indicates that both hydrophobic interactions and Ca+2 bridges or crosslinks may contribute to protein aggregation and micelle building.

Association of mixtures of the two major forms of beta-casein from human milk.

Human milk beta-casein (CN) is unique in that it may be phosphorylated at any level from zero (beta-CN-0P) to five (beta-CN-5P) organic phosphates per molecule. The 2P and 4P forms are the major components, with about 30 to 35% each. Here, we present the association properties of mixtures of these two moieties of human beta-CN. The aggregation patterns, as functions of temperature and ionic strength of these mixtures, generally follow those for the individual components. However, the mixtures yielded polymers with slightly different properties, which indicates extensive interaction between the two. Some properties of the mixtures were more like those for the 2P form, such as association in low salt buffer to give a peak with a sedimentation coefficient, s20,w, of approximately 11 S, in contrast to the 2P form alone with a peak of approximately 13 S and 4P alone with only a small amount of material with s20,w greater than 2 S at 27 degrees C. The solubility and interactions in the presence of Ca2+ ions were intermediate but more like the 4P form. A protein-concentration dependence for s20,w was seen, and laser light scattering indicated that there was an increase in size and/or a change in shape as the protein concentration increased. From the results, it is apparent that submicellar oligomers are probably formed by rapidly established equilibrium association reactions. The presence of an equal amount of the 2P form along with the 4P form does not appear to be a disadvantage in casein micelle formation and function.

Thermal cycling aids folding of a recombinant human beta-casein with four extra N-terminal amino acid residues.

Due to the limited secondary structure, it is believed that the caseins of milk, particularly the beta-caseins (beta-CN), may be in a mostly random-coil conformation or in various structures that result from random association of hydrophobic residues. However, the self-association of the human proteins with increasing temperature (T) and in the presence of Ca2+ is reproducible, implying that they normally fold into fixed tertiary structures. A nonphosphorylated recombinant human beta-CN with four extra amino acids at the N-terminus (GSHM-) was prepared and studied by laser light scattering, analytical ultracentrifugation, fluorescence spectroscopy, turbidity, and circular dichroism. In 3.3 M urea or at 4 degrees C, the protein was monomeric, as expected. Increasing T both without and with the addition of Ca2+ ions caused self-association as it does for the nonphosphorylated native beta-CN but with a somewhat different interaction pattern. However, returning the protein to its monomeric state by reequilibration at 4 degrees C followed again by increasing T caused a shift in the pattern. Such thermal cycling eventually caused the protein to equilibrate to a particular conformation where no more change could be observed. The resulting interaction pattern was similar to that of the native protein but differed particularly in that there was more extensive self-association for the recombinant mutant. The equilibration to a stable conformation was more rapid in the presence of Ca2+ ions. This suggests that the native protein normally folds into a particular conformation which may be aided by Ca2+ in the mammary gland. Further study of a recombinant form with the native amino acid sequence is needed.

Association of the quadruply phosphorylated beta-casein from human milk with the nonphosphorylated form.

Human beta-casein (beta-CN) is phosphorylated at levels from zero (beta-CN-0P) to five (beta-CN-5P). The major constituent is the 4P form (approximately 35%), whereas the 0P form (approximately 5%) has been implicated in the formation of a framework upon which the forms with higher levels of phosphorylation may aggregate. At 4 degrees C in 0.01 M imidazole and 0.02 M NaCl, pH 7, with a 1:1 (wt:wt) 0P:4P ratio and a total protein concentration of 3 mg/ml, the s20,w was 1.4 S (monomer). Laser light scattering gave a radius of approximately 4.5 nm. As the temperature, T, increased, s20,w increased to 2 S. At 25 degrees C, peaks of 9.5 S and 2 S were observed. This transition T was different from that of either form. At 37 degrees C, a single peak was again observed with s20,w of 17.5 S, compared with 42 S for the 0P and 14 S for the 4P form. Laser light scattering at 37 degrees C revealed a polymer of approximately 16 nm radius and D20,w of 1.55 cm2/s. A combination of D20,w and S20,w gave a relative molecular mass suggesting about 45 monomers per polymer. An incubation of 3 h or more at 37 degrees C caused further aggregation, characteristic of the 0P form, and supported the concept of framework formation. At pH 6.6, s20,w was 38 S compared with 1.4 S at pH 10.4. Hydrostatic pressure did not have a large effect but supported a soap micelle-like structure for the polymer. The turbidity of the mixture increased with the amount of CaCl2 and T until the protein precipitated. The properties of the 1:1 mixture of these human beta-CN are intermediate but probably more biased toward those for the 4P form.

Characterization of zinc-depleted alanyl-tRNA synthetase from Escherichia coli: role of zinc.

To evaluate the role of zinc in Escherichia coli alanyl-tRNA synthetase, hydrodynamic measurements and circular dichroism spectra were obtained for the zinc-depleted protein and compared with those of the native enzyme. At a protein concentration of 5 mg ml(-1), pH 7.5, the sedimentation coefficient (s(20,w)) was 6.3 S and was virtually independent of temperature between 10 and 37 degrees C, similar to the results reported for the native form. However, the s(20,w) now decreased significantly as the concentration increased, indicative of a possible change in conformation. The s(20,w) value did not appear to change as the pH was increased to 9.5. In standard buffer with 3.3 M added urea, a single peak with a s(20,w) of 3.6 S was obtained and with 6.6 M added urea, a peak with a s(20,w) of 2.7 S was seen. Added Gd-HCl (6 M) gave a single peak with s(20,w) of 2. 0 S. Like the native form, laser light scattering studies indicated some heterogeneity and a radius of 6.4 nm which was virtually independent of concentration and temperature in the range of 10-37 degrees C. At 25 degrees C, a diffusion coefficient (D(20,w)) of 3.3 x 10(-7) cm(2) s(-1) was obtained. The combination of s(0)(20,w) and D(20,w) yielded a molecular mass of approximately 179 kDa, which is slightly less than that reported for the native dimeric form (186 kDa). The intrinsic viscosity at 25 degrees C was extrapolated to 5. 3 ml g(-1), a value significantly higher than that reported for the native form, which increased with temperature. These results indicate some conformational and flexibility changes from the native to the zinc-depleted form, which may explain differences in activity. Furthermore, urea denaturation experiments demonstrate the role of zinc in stabilization of AlaRS structure.

Improvements in human health through production of human milk proteins in transgenic food plants.

Plants are particularly suitable bioreactors for the production of proteins, as their eukaryotic nature frequently directs the appropriate post-translational modifications of recombinant proteins to retain native biological activity. The autotrophic growth of plants makes this in vivo biosynthesis system economically competitive for supplementation or replacement of conventional production systems in the future. For the production of biologically active proteins, food plants provide the advantage of direct delivery via consumption of transformed plant tissues. Here we describe the production of recombinant human milk proteins in food plants for improvements in human nutrition and health, with emphasis on enhanced nutrition for non-breast fed infants as well as children and adults. Nutritional improvements in edible plants generated through advancements in recombinant DNA technology are rapidly repositioning the world for enjoyment of a more healthful diet for humans in all age groups.

The pH-dependent dissociation of beta-casein from human milk micelles: role of electrostatic interactions.

Evidence has been reported that supports the role of hydrophobic interactions in the association of the monomers and in the dissociation of different phosphorylation levels of beta-casein (CN) from human casein micelles. Here, the role of electrostatic interactions in the formation and structure of human casein micelles was examined by determining the beta-CN composition of micelle pellets from milk samples adjusted in the range from pH 5 to 10. As the pH was decreased from normal (approximately pH 7.5) at 37 degrees C, the proteins remained associated with the micelle, and the relative amounts of all of the phosphorylated forms remained essentially constant. As the pH was increased from normal, protein was lost from the micelles to the supernate. When the relative micelle compositions were corrected for the loss, all of the phosphorylated forms decreased in total amount: the largest decrease for both the nonphosphorylated and singly phosphorylated forms (approximately 95%), a sizeable decrease for the doubly phosphorylated form (approximately 70%), and only about a 25% decrease for the triply, quadruply, and fully phosphorylated forms. Laser light scattering measurements on micelle pellets resuspended in simulated milk ultrafiltrate at 37 degrees C yielded mean particle radii of approximately 63 nm at pH 7.5 and approximately 35 nm near pH 6. These results suggest that micelle stabilization depends not only on the net charge on the negatively charged organic phosphate esters and the inorganic orthophosphate, which vary with pH and calcium binding, but also on the positively charged amino groups of basic amino acid residues in the proteins.

Monomer characterization and studies of self-association of the major beta-casein of human milk.

The casein form that has four organic phosphoryl groups, beta-casein (CN)-4P, is the major constituent (approximately 35%) of the beta-CN fraction of human milk and should play an important role in micelle structure and formation. In 3.3 M urea, the monomer is present with a molecular mass of 24,500 Da and a sedimentation coefficient of 1.3 S (Svedberg units, 10(-13)s). In 0.02 M NaCl and 0.01 M imidazole (low salt buffer) at pH 7, the sedimentation coefficient was 1.5 S, which increased to 14 S at 37 degrees C. Laser light scattering in low salt buffer and 9 mg/ml of protein indicated monomers with a radius of about 4 nm at 4 degrees C. The size of the radius increased as temperature increased, and, at 37 degrees C, the radius was about 12 nm. The molecular mass suggested the presence of about 47 monomers per polymer. In 0.25 M NaCl and with 10 nM Ca2+ prior to precipitation, the polymer attained a maximum radius of about 15 nm, which perhaps is the size of the smallest human milk micelles. The low value for reduced viscosity of 8.2 ml/g for the calcium-induced polymer was independent of protein concentration, suggesting a spherical shape and fixed size. Calcium apparently binds strongly to the phosphates; the dissociation constant was 8.1 x 10(-4) M. Other constituents of milk, such as inorganic orthophosphate, may contribute to differences in the manner by which beta-CN, with various phosphorylation levels, participate in micelle formation.

Expression of the human milk protein beta-casein in transgenic potato plants.

A 1177 bp cDNA fragment encoding the human milk protein beta-casein was introduced into Solanum tuberosum cells under control of the auxin-inducible, bidirectional mannopine synthase (mas1',2') promoters using Agrobacterium tumefaciens-mediated leaf disc transformation methods. Antibiotic-resistant plants were regenerated and transformants selected based on luciferase activity carried by the expression vector containing the human beta-casein cDNA. The presence of human beta-casein cDNA in the plant genome was detected by PCR and DNA hybridization experiments. Human beta-casein mRNA was identified in leaf tissues of transgenic plants by RT-PCR analysis. Human beta-casein was identified in auxin-induced leaf and tuber tissues of transformed potato plants by immunoprecipitation and immunoblot analysis. Human beta-casein produced in transgenic plants migrated in polyacrylamide gels as a single band with an approximate molecular mass of 30 kDa. Immunoblot experiments identified approximately 0.01% of the total soluble protein of transgenic potato leaf tissue as beta-casein. The above experiments demonstrate the expression of human milk beta-casein as part of an edible food plant. These findings open the way for reconstitution of human milk in edible plants for replacement of bovine milk in baby foods for general improvement of infant nutrition, and for prevention of gastric and intestinal diseases in children.

Structural studies on casein micelles of human milk: dissociation of beta-casein of different phosphorylation levels induced by cooling and ethylenediaminetetraacetate.

Information on the structure of human casein micelles has been obtained from dissociation of beta-casein (CN). Two approaches were used: cooling at 4 degrees C and addition of EDTA. An initial loss of about 80% of the protein optical density occurred upon cooling to 4 degrees C. Dissociation was time dependent, and at > or = 24 h about 10% remained. However, mean size and voluminosity of micelles increased, as indicated by laser light scattering and viscosity measurements. This process was reversible, and 95% of the protein reentered the micelles upon incubation for 3 h at 37 degrees C. Upon cooling, amounts of nonphosphorylated beta-CN increased, and singly phosphorylated beta-CN levels were almost constant relative to the total beta-CN in micelles. Upon addition of EDTA (0 to 5 mM), the forms with three to five phosphates were the major dissociating constituents; EDTA that was added by dialysis produced similar results but at lower concentrations. These data suggest that, in the absence of significant amounts of alpha s1-CN, nonphosphorylated and singly phosphorylated human beta-CN may form a framework, as proposed for alpha s1-CN for bovine milk, along with the colloidal calcium phosphate for the development of the final micelle structure by addition of the more highly phosphorylated forms. The results also indicate that human casein micelles have a less rigid structure than those of other species.

Further characterization of Escherichia coli alanyl-tRNA synthetase.

Selected physical and thermodynamic parameters for Escherichia coli alanyl-tRNA synthetase (AlaRS) have been determined primarily to assess the quaternary structure of this enzyme. The extinction coefficient (epsilon) at 280 nm was determined experimentally to be 0.71 ml mg-1 cm-1, and the partial specific volume (nu) was calculated from the amino acid composition to be 0.73 ml g-1. From viscosity experiments the intrinsic viscosity (eta) of AlaRS was extrapolated to be 3.4 ml g-1 and the degree of hydration (delta 1) estimated to be 0.67 gH2O g(-1)(AlaRS). Laser light-scattering studies indicated some heterogeneity; a radius of 6.3 nm was calculated for the major fraction with a diffusion coefficient (D20,W) of 3.89 x 10(-7) cm2 s-1. In 50 mM Hepes, pH 7.5, 20 mM KCl, 2 mM 2-mercaptoethanol and at a protein concentration of 4.2 mg ml-1 the sedimentation coefficient (S20,W) was 6.36 S; this value increased slightly when the protein concentration was decreased. The combination of S20,W and D20,W under these conditions yielded a molecular weight of approximately 186,000 Da, corresponding to a dimer. The S20,W was virtually independent of temperature in the range of 10-37 degrees C, while an Arrhenius plot of aminoacylation activity was biphasic. The isoelectric point was determined experimentally to be 4.9. Sedimentation equilibrium data were best fit to a decamer association complex in which dimeric AlaRS is the predominant species at 25 degrees C.

Kappa-casein and beta-caseins in human milk micelles: structural studies.

The function of human milk micelles is to deliver nutrients, particularly insoluble minerals such as the necessary calcium phosphate, to the child in a readily ingested suspension that meets the special requirements of the human infant digestive system. The micelle structure which fulfills that function is not known. However, the development of ion-exchange and reverse-phase HPLC methods for the purification and quantitation of the kappa- and beta-caseins, along with tritium labeling of the carbohydrate of the kappa-casein in human milk to aid in its detection, provided the tools for probing micelle structure by examining the composition of micelles fractionated according to size by differential centrifugation. The relative amount of kappa-casein increased as micelle size decreased, and thus as surface area/volume increased. Since kappa-casein also stabilizes the micelles against precipitation by Ca2+ ions, a surface position for most of the kappa-casein is implied. The relative amounts of the various phosphorylation levels of the human beta-caseins remained essentially constant except for the nonphosphorylated (O-P) form, which apparently decreased as micelle size decreased. These data suggest that the beta-caseins are linked in the micelles partially through electrostatic interactions involving the organic phosphoryl groups, lacking in the O-P, so that O-P can dissociate into the whey. That further implies that the complete surface is not protected by the kappa -casein but that the beta-caseins are also accessible to the solution.

Interaction properties of singly phosphorylated human beta-casein: similarities with other phosphorylation levels.

The singly and doubly phosphorylated forms of the human beta-caseins may function differently in casein micelle formation in human milk than the forms with higher phosphorylation (levels 3 to 5). This paper reports properties of the singly phosphorylated protein and compares them with the same previously measured properties for the forms with 0, 2, 3, and 5 phosphoryl groups, including the monomer characteristics, such as the extinction coefficient, partial specific volume, and molecular weight; the Ca2+ binding; the self-association and solubility patterns as monitored visually and with laser light scattering, sedimentation velocity, intrinsic viscosity, and ultraviolet spectroscopy; and the fluorescence polarization and intensity that are due to the binding of a fluorescent probe and to environmental changes in the vicinity of an intrinsic fluorophore. Changes could be attributed to relatively minor electrostatic differences between entities that vary in phosphoryl content. Major differences in interaction patterns between phosphorylation levels must therefore reside in the more complicated system in which inorganic o-phosphate is also present, thus permitting the proteins to interact with and be crosslinked by colloidal calcium phosphate.

Characterization of human kappa-casein purified by FPLC.

Because previous purification procedures for human kappa-casein may have caused the loss of some carbohydrate, relatively gentle methods were used. The protein was isolated by a four-step procedure which included isoelectric precipitation of whole casein, gel chromatography on Sephadex G-200 in the presence of SDS, removal of the SDS with Extracti-Gel D, and FPLC chromatography on Mono Q with buffers containing 6 M urea. The purified protein was nearly identical in amino acid composition to that found earlier by amino acid analysis and peptide sequencing and a molar extinction coefficient of 11.2 +/- 0.1 was determined on the basis of amino acid analysis with a norleucine internal standard. Hydrolysis, acylation, and methylsilylation of the carbohydrate, followed by gas chromatographic analysis on a fused silica column, yielded approximately 5% fucose, 17% galactose, 18% N-acetylglucosamine, 8% N-acetylgalactosamine and 7% sialic acid, totaling almost 55% by weight. The percentages from two different donors were almost the same. About 1 mole phosphorus per mole of kappa-casein was also detected. Using low-speed sedimentation equilibrium methods, a molecular weight of only 33,400 was obtained for human kappa-casein, suggesting carbohydrate lability. Human beta-casein with four phosphoryls was stabilized against precipitation by 10 mM Ca+2 ions at a level greater than 95% when the molar ratio of kappa/beta exceeded 0.15.

Interaction properties of doubly phosphorylated beta-casein, a major component of the human milk caseins.

Doubly phosphorylated beta-casein constitutes nearly 30% of the total human beta-caseins and is thus one of the major components of that fraction. The properties and mode of association of doubly phosphorylated beta-casein are therefore important determinants of the structure and function of the human casein micelle. Doubly phosphorylated beta-casein has an absorbency of 6.2 and a partial specific volume of .74. The protein precipitated at room temperature when 10 mM Ca2+ was added but produced a clear solution in 1 M NaCl. Equilibrium dialysis produced an average of 2.06 major Ca(2+)-binding sites at 37 degrees C with a dissociation constant of 12.1 x 10(-4) M. The monomer at 20 degrees C was calculated to have a solvation of 2.1 g of H2O/g of protein and an axial ratio of 6.8, suggesting a prolate ellipsoid of about 11 by 2 nm. At high ionic strength, evidence exists for a spherical structure with a molecular weight of 2.25 x 10(6). This structure would represent a polymer of about 90 monomers with a radius of 14.8 nm and a solvation of 1.93 g of H2O/g of protein. This association behavior is similar to that of other phosphorylated human beta-caseins but differs from the nonphosphorylated form. It changes when both Ca2+ and inorganic orthophosphate are present.

Interactions of triply phosphorylated human beta-casein: fluorescence spectroscopy and light-scattering studies of conformation and self-association.

Structural changes of triply phosphorylated human beta-casein, caused by shifts in temperature between 5 and 40 degrees C, were studied using intrinsic and extrinsic fluorescence, fluorescence polarization, turbidity, and light scattering measurements. Intrinsic fluorescence declined between 5 and 20 degrees C then rose between 25 and 40 degrees C, indicative of a shift of the tryptophan fluor toward a more nonpolar environment. The fluorescence of the extrinsic probe, 8-anilino-1-naphthalene-sulfonic acid (ANS), increased only slightly between 5 and 25 degrees C, and then more sharply between 25 and 40 degrees C, suggesting a change in conformation leading to a change in either the dissociation constant, Kd, or the number of ANS binding sites, N. The presence of Ca+2 ions did not significantly alter the pattern of changes of intrinsic and extrinsic fluorescence with changing temperature. For ANS binding, values of Kd and N were calculated by two different procedures, each based upon different assumptions. The results point to increased exposure of hydrophobic surfaces with increased temperature, strongly supportive of conformational changes. Although more opportunity for hydrophobic interaction leads to increased protein-protein association, turbidity and light-scattering also suggest ion bridge formation between protein molecules. A comparison of the primary sequences of beta-caseins from six species reveal residues that are common in all species examined and thus are pivotal in protein folding and conformation, intermolecular hydrophobic interactions and ion bridge formation with Ca+2 and inorganic phosphate.

Choline inhibition of prothrombin activation.

A computer-interfaced spectrophotometric kinetic assay for prothrombin activation was developed, coupling the production of thrombin to a thrombin-specific amidolytic chromogenic reaction. As thrombin accumulated initially at constant velocity, the simultaneous release from S-2238 of pNA conformed to an acceleration function. Adherence to the acceleration function of the temporally increasing A400 of pNA was evaluated after transforming the data into linear format which permitted linear regression analysis. High correlation coefficients, routinely greater than 0.99, verified linearity of thrombin production in individual assay mixtures. As prothrombin concentrations were varied, factor Xa exhibited Michaelis-Menten kinetics. Added choline produced a pattern of mixed-type inhibition. Replots of LB slopes and intercepts versus choline concentration gave apparent Ki and Ki' values (mM): 22 +/- 3 and 48 +/- 7 without factor Va, 25 +/- 4 and 41 +/- 4 with factor Va.